Sir2 products and activities

ABSTRACT

A novel compound, 2′/3′-O-acetyl-ADP-ribose, is provided. The compound is a mixture of the 2′ and 3′ regioisomers of O-acetyl-ADP ribose, and is formed nonenzymatically from 2′-O-acetyl-ADP-ribose, which is the newly discovered product of the reaction of Sir2 enzymes with acetylated peptides and NAD + . Analogs of 2′/3′-O-acetyl-ADP-ribose are also provided. Additionally, methods of preparing 2′/3′-O-acetyl-ADP-ribose, methods of determining whether a test compound is an inhibitor of a Sir2 enzyme, methods of detecting Sir2 activity in a composition, methods of deacetylating an acetylated peptide, and methods of inhibiting the deacetylation of an acetylated peptide are provided. Prodrugs of 2′/3′-O-acetyl-ADP-ribose are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/331,919, filed Nov. 21, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. Government support under NationalInstitutes of Health Grant No. AI 34342. The Government has certainrights to the invention.

BACKGROUND

(1) Field of the Invention

The present invention generally relates to enzyme products andactivities. More particularly, the invention is directed to thediscovery of products and activities of Sir2 enzymes.

(2) Description of the Related Art

References Cited

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The Sir2p-like enzymes are broadly conserved from bacteria tohumans^(1,2). In yeast, these proteins form complexes with otherproteins to silence chromatin³⁻⁶ by accessing histones^(7,8) anddeacetylating them⁹⁻¹². Sir2 enzymes are homologs of the bacterialenzyme cobB, a phosphoribosyltransferase¹³, which led to the findingthat Sir2p employs NAD⁺ as a co-substrate in deacetylationreactions^(10,14,12). This unusual requirement for NAD⁺ isstoichiometric¹⁴ and generates a novel product originally proposed to beβ-1′-AADPR^(15,16) or possibly 2′-AADPR^(16,17). A crystal structure ofa Sir2p homolog from Archaeoglobus fulgidus called Sir2-Af1 was recentlydetermined with NAD⁺ bound at the active site. The structure wasinterpreted in the context of a catalytic mechanism that producesβ-1′-AADPR¹⁸. A cleft is proposed to bind the acetyl-lysine side chainof substrate proteins in proximity to the C1′ of the bound NAD⁺,providing substrate organization for acetyl group transfer between thepeptide side chain and NAD⁺¹⁸.

From mechanistic and thermodynamic considerations, the NAD⁺ dependentdeacetylation by Sir2p is an unusual reaction, since Lys N-deacetylationreactions are simple to accomplish by hydrolysis alone. The apparentcoupling of hydrolysis and ADP-ribose transfer to acetate formsacetyl-ADP-ribose (AADPR) as product, and generates a new metabolite ofunknown function¹⁵⁻¹⁷.

Evidence to date indicates that histones are in vivo substrates of Sir2enzymes. Yet, the genomes of eubacteria such as Salmonella which lackhistones, encode Sir2-like proteins¹⁹. Similarly, archaeal genomes thatencode Sir2ps also encode histone-like proteins, but those histones lackthe N-terminal tails that are the prominent sites of lysine acetylationin eukaryotic histones. One archaeal species, Archaeoglobus fulgidus,encodes two different Sir2-like deacetylases that are 47% identical andthe X-ray structure of one of these (Sir2Af1) was recently determined¹⁸.Sir2Af2 has been characterized more extensively biochemically, and isactive in vitro on defined substrates¹².

Histone deacetylation by Sir2p is proposed to be the major reaction thatconverts chromatin from active to silent states^(9,10,12). Thissuggestion is supported by studies of silencing complexes which showSir2p protein is the only conserved SIR family member in all yeastsilencing complexes studied²⁸. Caloric restriction upregulates Sir2pactivity and may extend lifespan, showing that silencing might havepotent biological effects in various organisms^(29,30). The requirementfor NAD⁺ and evidence that silencing factors can “sense” the redox stateof the cell suggests that Sir2p family members are uniqueamide-hydrolysis enzymes with broad roles^(12,31).

The distribution of Sir2p family of enzymes into organisms withouthistone substrates, and eukaryotic genomes encoding multiple Sir2proteins, suggest a family of deacetylases with varying substrates.Mutagenesis experiments suggest that the N- and C-terminal regionsflanking the catalytic core domain of Sir2p help direct it to differenttargets³². Although most Sir2 proteins in eukaryotic cells are locatedin the nucleus³³ others are cytosolic³⁴⁻³⁶, or even mitochondrial(Onyango, Celic, Boeke and Feinberg; unpublished observations) whichsuggests additional substrates.

SUMMARY OF THE INVENTION

The present invention is based on two discoveries. The first discoveryis the determination that a product of the reaction of a Sir2 with anacetylated peptide and NAD⁺ is 2′-O-acetyl-ADP-ribose. The 2′ acetylgroup can isomerize with the 3′ hydroxyl, with the reverse isomerizationoccurring more slowly, until an equilibrium is formed between the 2′ and3′ forms. Thus, the Sir2 reaction ultimately causes the production of2′/3′-O-acetyl-ADP-ribose (FIG. 10).

The second discovery is that Sir2 enzymes deacetylate peptides otherthan histones. Examples of such peptides are acetylated p53 andfragments thereof. Based on experimental results described in Example 1,the skilled artisan would expect that Sir2 enzymes deacetylate anyacetylated peptide of at least two amino acids, wherein at least one ofthe amino acids comprises a lysine residue that is acetylated at theε-amino moiety.

Accordingly, the present invention is directed to purified preparationsof 2′/3′-O-acetyl-ADP-ribose, and analogs thereof that exhibit increasedstability in cells.

The present invention is also directed to methods of preparing2′/3′-O-acetyl-ADP-ribose. The methods comprise combining a Sir2 enzymewith NAD⁺ and an acetylated peptide substrate of Sir2 in a reactionmixture under conditions and for a time sufficient to deacetylate thepeptide, then identifying and purifying the 2′/3′-O-acetyl-ADP-ribosefrom the reaction mixture.

Additionally, the present invention is directed to methods ofdetermining whether a test compound is an inhibitor of a Sir2 enzyme.The methods comprise combining the test compound with the Sir2 enzyme,NAD⁺ and an acetylated peptide substrate of Sir2 under conditions andfor a time sufficient to deacetylate the peptide in the absence of thetest compound, quantifying 2′/3′-O-acetyl-ADP-ribose produced from theacetylated peptide, then comparing the quantity of2′/3′-O-acetyl-ADP-ribose produced with a quantity of2′/3′-O-acetyl-ADP-ribose produced under the same conditions but withoutthe test compound. In these methods, the presence of less2′-O-acetyl-ADP-ribose and/or 3′-O-acetyl-ADP-ribose than without thetest compound indicates that the test compound is an inhibitor of theSir2 enzyme.

In other embodiments, the present invention is directed to methods ofdetecting activity of a Sir2 in a composition. The methods comprisecombining the composition with NAD⁺ and an acetylated peptide substrateof a Sir2 under conditions and for a time sufficient to deacetylate thepeptide in the presence of Sir2 activity, then measuring2′/3′-O-acetyl-ADP-ribose. The presence of 2′/3′-O-acetyl-ADP-riboseindicates the presence of Sir2 activity in the composition.

The present invention is additionally directed to methods ofdeacetylating an acetylated peptide. The methods comprise combining thepeptide with a Sir2 enzyme. In novel aspects of these embodiments, theacetylated peptide is not a histone.

Additionally, the present invention is directed to methods of inhibitingthe deacetylation of an acetylated peptide. The methods comprise mixingthe acetylated peptide with 2′/3′-O-acetyl-ADP-ribose or an analogthereof.

In additional embodiments, the invention is directed to prodrugs of2′/3′-O-acetyl-ADP-ribose. The prodrugs comprise2′/3′-O-acetyl-ADP-ribose covalently bonded to a moiety by anesterase-sensitive bond. In these embodiments, the prodrug is capable ofpassing into a cell more easily than 2′/3′-O-acetyl-ADP-ribose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphics, HPLC traces and immunoblots of p53 and derivedpeptides are substrates for Sir2p and related proteins. A. Boldedresidues are acetyl-lysine in JB11 and 12 and lysine in JB12D. B–G. HPLCseparation (numbers are retention times) and MS analysis (numbers areamu) of substrate and product peaks. B. HPLC analysis of JB11 reactionwith Sir2p. C, D. MS of two major peaks (product, C; substrate, D) fromHPLC in panel B. E. HPLC of peptide JB12. F. HPLC of peptide JB12following treatment with Sir2p. Inset is MS of major peak. G. HPLC ofmixture of peptides JB12 and JB12D. H. p53 activity on full-length p53expressed in yeast, immunoprecipitated with anti-p53 DO-1 and acetylatedin vitro with p300. Upper panel is immunoblotted with anti-acetyl p53antibody specific to lysines 373 and 382. Lower panel is immunoblottedwith antibody FL-393 reactive against acetylated and deacetylated formsof p53.

FIG. 2 shows NMR traces of various preparations relating to thestructure of the SIR2 product. Top Panel: ¹H NMR of β-1′-AADPR in D₂O(300 MHz). Middle Panel: ¹H NMR of 3′-AADPR at 15° C. in D₂O (600 MHz).Bottom Panel: Sample from middle panel aged several h at 20° C. (600MHz) showing equilibrium of 2′- and 3′-AADPR isomers.

FIG. 3 shows an MS/MS comparison of Sir2p derived AADPR and β-1′-AADPR.

FIG. 4 shows plots and a chromatogram relating to the structure ofAADPR. Upper left panel: Time dependence of HPLC chromatograms showingconversion of 3′-AADPR to 2′-AADPR isomer over 5 h at 15° C. Lowerpanel: Stack plot of 1D ¹H NMR spectra showing changes in 3′ (α and β)AADPR (major) and 2′ (α and β) AADPR (minor) regioisomer populations asa function of time at 10° C. Upper right panel: Plot of 3′ O-acetyl-ADPRisomer as a function of time at 15° C. as measured by HPLC. Solid curverepresents best fit to the first order function described in the text.The dotted line represents the equilibrium determined by integration ofHPLC peak areas after 24 h incubation.

FIG. 5 shows a stack plot of 1D ¹H NMR spectra (600 MHz) taken at 5° C.,showing initial formation of 2′-AADPR (resonances at d 5.15 (2′β), and4.93 (2′α)) catalyzed by 50 mM Sir2Af2 in the presence of 10 mM NAD⁺,and 1.5 mM JB12 peptide. Later time points illustrate conversion of2′-ADPR to 3′-ADPR (resonances at d 5.04 (3′β), and 4.88 (3′α)). Spectraare separated in time by 1.0 hour. Inset: MS spectra of molecular ionsof AADPR derived from Sir2Af2 reactions after treatment with isotopelabeled water in reaction and/or in quench. Top panel shows ions derivedfrom a Sir2Af2 reaction in ¹⁸O water followed by a H₂ ¹⁸O quenchcontaining 10% d₄-acetic acid for 3 h at room temperature. Middle panelshows ions derived from the Sir2Af2 reaction in ¹⁸O water withoutquenching. Bottom panel shows ions derived from the Sir2Af2 reaction inunlabeled water without quenching. Results for other conditions andexperimental details are listed in Table 2 and Table 2 legend.

FIG. 6 shows two graphics showing the acetyl-lysine side-chain, dockedinto the active site cavity of Sir2Af118. Left and right panels arepeptide backbone and space filling models, respectively. Theacetyl-oxygen from the N-acetyl-lysine at residue 382 is within van derWaals contact of C1′ of NAD⁺. The acetyl moiety can be placed in theproper attack geometry to form an acyl-O-C1′ bond with astereochemistry. The p53 C-terminal peptide containing N-acetylatedlysine 382 is a schematic model only.

FIG. 7 shows two alternative deacetylation mechanisms proposed for SIR2(A) Deacetylation mechanism initiated by 2′-OH nucleophilic attack ofthe amide carbonyl of an acetyl lysine residue. Mechanism A is unlikelybecause mutation of His to Ala would eliminate nicotinamide exchange.His116 ligated to 3′-OH acts as a base catalyst to activate 2′-OHthrough a proton-transfer relay mechanism via 3′-OH. (B) Deacetylationmechanism dependent upon the breakdown of 1′-O-amidate by 2′-OH attack,downstream from nicotinamide bond cleavage. Mechanism B permitsnicotinamide exchange prior to 2′-OH activation, consitent with theexchange reported for the His to Ala mutant.

FIG. 8 shows varius reaction schemes discussed in the Specification.

-   Scheme 1A. CD38 catalyzed synthesis of β-1′-AADPR.-   Scheme 1B. Observed equilibria of 2′-substituted and 3′- substituted    α and β AADPRs and relative equilibrium populations at 15° C.-   Scheme 1C. Reaction sequence from NAD⁺ in Sir2p catalyzed    deacetylations.-   Scheme 1D. ADP-ribosylation dependent deacetylation generating    a-1′-AADPR with migration to 2′-AADPR.-   Scheme 1E. ADP-ribosylation dependent Sir2p catalyzed deacetylation    mechanism generating 2′-AADPR, 3′-AADPR and ¹⁸O labeled products.

FIG. 9 shows an NMR trace of 2′/3′-O-AADPR.

FIG. 10 shows the structure of 2′/3′-O-AADPR.

FIG. 11 shows the separation of NAD⁺ and AADPR to measure Sir2 activityin different time incubations of Sir2 with NAD⁺ and peptide JB12.Minimum incubation time is 30 minutes.

FIG. 12 is a graph showing Sir2 mediated AADPR production versus time asmeasured by column-isolated AADPR and radioactivity assay.

FIG. 13 is a graph showing para-nitrophenyl acetate measured esteraseactivity in a DEAE-separated liver extract (no I), compared topara-nitrophenyl acetate measured esterase activity in the presence ofmM concentrations of an AADPR-like Sir2 inhibitor compound (I).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “AADPR” means acetyl adenosine diphosphate ribose. 1′-,2′, and 3′-AADPR means the 1′-O, 2′-O and 3′-O regioisomers,respectively, of AADPR. “2′/3′-AADPR” or “2′/3′-O-AADPR” means a mixtureof the 2′-AADPR and 3′-AADPR (FIG. 10). “ADPR” means adenosinediphosphate ribose.

As used herein, “Sir2” means silent information regulator 2, or any ofits orthologs or paralogs, now known or later discovered, as would beunderstood by the skilled artisan.

As used herein, “HPLC” means high performance liquid chromatorgraphy;“NMR” means nuclear magnetic resonance; and “MS” means mass spectroscopyor mass spectrometer.

As used herein, “transfection” is the process of inserting DNA into acell such that genes encoded by the DNA are capable of being expressedby the cell. The DNA can be transiently expressed or stably expressed inthe cell.

As used herein, a “peptide” is a sequence of at least two amino acids.Peptides can consist of short as well as long amino acid sequences,including proteins.

As used herein, “NAD⁺” means nicotinamide adenine dinucleotide.

The present invention is based on two discoveries. The first discoveryis the determination that a product of the reaction of a Sir2 with anacetylated peptide and NAD⁺ is 2′-O-acetyl-ADP-ribose. The 2′ acetylgroup can transfer to the 3′ hydroxyl until an equilibrium is formedbetween the 2′ and 3′ forms. Thus, the Sir2 reaction ultimately causesthe production of 2′/3′-O-acetyl-ADP-ribose (“2′/3′-AADPR”-FIG. 10).This is contrary to the previously held belief that the product of thereaction is β-1′-AADPR^(15,16). The 2′/3′-AADPR has not been previouslydescribed. Because Sir2 uses metabolically valuable NAD⁺ to produce2′/3′-AADPR, and because the product 2′/3′-AADPR is metabolicallyunstable, the skilled artisan would understand that the product islikely useful as initiators of signaling pathways. The 2′/3′-AADPRproduct is also useful for, inter alia, testing for inhibitors of Sir2enzymes, for determining and quantifying Sir2 activity in a composition,and for inhibiting Sir2 enzymes. These uses for 2′/3′-AADPR are furtherdescribed below.

The second discovery that leads to the present invention is that Sir2enzymes deacetylate peptides other than histones. Examples of suchpeptides are acetylated p53 and fragments thereof. Based on experimentalresults described in the Example, the skilled artisan would expect thatSir2 enzymes deacetylate any acetylated peptide of at least two aminoacids, wherein at least one of the amino acids comprises a lysineresidue that is acetylated at the ε-amino moiety. This finding wouldlead the skilled artisan to believe that Sir2 enzymes have a muchbroader role in regulating transcription than was previouslyappreciated, since it is now understood that Sir2 enzymes candeacetylate any acetylated protein.

Accordingly, the present invention is directed to a purified preparationof 2′/3′-O-acetyl-ADP-ribose. As used herein, “purified” means occupyinga greater proportion of a composition than would be found in nature.Preferably, the purified preparation is an aqueous composition having asolute (excluding salts) that is at least 50% 2′/3′-AADPR. Morepreferably, the purified preparation is at least 75% 2′/3′-AADPR. Ineven more preferred embodiments, the purified preparation is at least90% 2′/3′-AADPR. The purity of the preparation can be determined by anymeans known in the art. A preferred method for determining the purity of2′/3′-AADPR is by HPLC, e.g., by methods described in the Example below.

In a living cell or a cellular or tissue extract, the 2′/3′-AADPR isgenerally very short-lived, unless the action of endogenous esterasesare arrested. This ephemeral quality of 2′/3′-AADPR can be circumventedby using analogs of 2′-AADPR or 3′-AADPR that are designed to haveincreased stability from esterase action through the use of well-knownsubstitutes for ester oxygen atoms that are subject to esterase attack.The esterase-labile oxygen atoms in 2′-AADPR and 3′-AADPR would beunderstood to be the ester oxygen linking the acetate group with theribose, and the ester oxygen between the two phosphorus atoms. As isknown in the art, substitution of either or both of these ester oxygenatoms with a CF₂, a NH, or a S would be expected to provide a 2′-AADPRor 3′-AADPR analog that is substantially more stable due to increasedresistance to esterase action.

Thus, in some embodiments, the invention is directed to analogs2′-O-acetyl-ADP-ribose or 3′-O-acetyl-ADP-ribose exhibiting increasedstability in cells. The preferred analogs comprise a CF₂, a NH, or a Sinstead of the acetyl ester oxygen or the oxygen between two phosphorusatoms. The most preferred substitute is CF₂. Replacement of the acetylester oxygen is particularly preferred. In other preferred embodiments,both the ester oxygen and the oxygen between the two phosphorus atomsare independently substituted with a CF₂, a NH, or a S.

The 2′/3′-O-acetyl-ADP-ribose product can be prepared by any of a numberof chemical or enzymatic methods in the art. Preferably, the2′/3′-O-acetyl-ADP-ribose is prepared using a Sir2 enzyme, by combiningthe Sir2 enzyme with NAD⁺ and an acetylated peptide substrate of theSir2 in a reaction mixture under conditions and for a time sufficient todeacetylate the peptide. The 2′/3′-O-acetyl-ADP-ribose can be identifiedand purified from the reaction mixture. Preferably, the2′/3′-O-acetyl-ADP-ribose is purified chromatographically, for exampleby HPLC. In some embodiments, the isolation and/or quantitation of2′/3′-O-acetyl-ADP-ribose can be advantageously simplified by usingradiolabeled NAD⁺ such that the 2′/3′-O-acetyl-ADP-ribose isradiolabeled after the peptide is deacetylated. See, e.g., Example 2 fora non-limiting example of those methods. Alternatively, the skilledartisan would understand that a radioactive acetate-labeled acetylatedpeptide would also label 2′/3′-O-acetyl-ADP-ribose. The labeled2′/3′-O-acetyl-ADP-ribose allows for simpler separation methods, wherethe labeled 2′/3′-O-acetyl-ADP-ribose need only be separated from theradiolabeled NAD⁺ or acetylated peptide substrate, e.g., with aSephadex-DEAE column (see, e.g., Example 2 and FIG. 11). Once separatedfrom the radiolabeled substrate(s), radiolabeled2′/3′-O-acetyl-ADP-ribose can be easily quantified, e.g., byscintillation, which is useful for methods described below wherequantitation of Sir2, or determination of inhibitor activity, isdesired.

As previously discussed, it is believed that any acetylated peptide ofat least two amino acids can usefully serve as a substrate for Sir2,provided at least one of the amino acids is a lysine residue that isacetylated at the ε-amino moiety. The acetylated peptide can compriseany number of amino acids, including three, five, ten, fifteen,eighteen, or more amino acid residues. As is known, there is noparticular sequence that is preferred, although most deacetylationoccurs in a pair of basic amino acids. See, e.g., ref. 10 and Example 1.

In preferred embodiments, the acetylated peptide substrate is homologousto an acetylated region of the regulatory domain of a p53, for examplepeptide JB11 or JB12, described in the Example. An acetylated p53 isalso a useful substrate for Sir2.

In other embodiments, the acetylated peptide substrate is homologous toan acetylated region of a histone, or an acetylated histone itself.

The described enzyme reaction is expected to be for any Sir2 enzyme,with the exception of Sir2Af1 (see Example). Well-known Sir2 enzymesthat would catalyze the described reaction includes, inter alia,Sir2Af2, human Sir2A, yeast Sir2p, Sir2Tm from Thermotoga maritima, andcobB, from Salmonella typhimurium. Useful enzymes include those derivedfrom prokaryotes, including archaeal bacteria and eubacteria, and thosederived from prokaryotes, including yeast and humans.

As used herein, the term “derived from”, when referring to a Sir2enzyme, means either (a) extracted from the organism that naturallyproduces the enzyme, or (b) translated, in vitro or in vivo, from a geneencoding the Sir2 enzyme from the organism. This includes genestransfected into heterologous organisms, e.g., a Sir2 gene derived froma human, translated from the human Sir2 gene transfected into E. coli,yeast, a mammalian cell, or an intact mammal.

In additional embodiments, the present invention is directed to methodsfor determining whether a test compound is an inhibitor of a Sir2enzyme. The methods comprise the following steps:

-   -   combining the test compound with the Sir2 enzyme, NAD⁺ and an        acetylated peptide substrate of Sir2 in a reaction mixture under        conditions and for a time sufficient to deacetylate the peptide        in the absence of the test compound;    -   quantifying 2′/3′-O-acetyl-ADP-ribose produced from the        acetylated peptide; then    -   comparing the quantity of 2′/3′-O-acetyl-ADP-ribose produced        with a quantity of 2′/3′-O-acetyl-ADP-ribose produced under the        same conditions but without the test compound. In these methods,        the presence of less 2′-O-acetyl-ADP-ribose and/or        3′-O-acetyl-ADP-ribose with the test compound than without the        test compound indicates that the test compound is an inhibitor        of the Sir2 enzyme.

These methods are useful for evaluating potential inhibitors with anySir2 enzyme that normally produces 2′/3′-O-acetyl-ADP-ribose. Peptidesuseful for these reactions include any of the Sir2 peptide substratespreviously described. As previously discussed, using a radiolabeled Sir2substrate can simplify these methods.

In related embodiments, the invention is directed to methods ofinhibiting Sir2 enzymes. The methods comprise combining the Sir2 enzymewith an inhibitor found by the method described directly above. In thesemethods, any Sir2 enzyme that normally produces2′/3′-O-acetyl-ADP-ribose is a useful target. The Sir2 enzyme to beinhibited can be within a living cell, wherein the inhibitor is insertedinto the cell by any of a number of methods, depending on the chemicalcharacteristics of the inhibitor, as is know in the art. For example,the living cell can be a bacterial cell or eukaryotic cell, such as ayeast or a mammalian cell, including a human cell. The mammalian cellcan be within a living mammal. In some useful embodiments, the mammal isat risk for, or suffering from, cancer induced by lack of p53 -DNAbinding. In those embodiments, the Sir2 inhibitor would be expected toprevent Sir2 from deacetylating p53, thus increasing p53 DNA binding.

The present invention is also directed to methods of detecting activityof a Sir2 in a composition. The methods comprise the following steps:

-   -   combining the composition with NAD⁺ and an acetylated peptide        substrate of a Sir2 under conditions and for a time sufficient        to deacetylate the peptide in the presence of Sir2 activity;        then    -   measuring 2′/3′-O-acetyl-ADP-ribose. In these embodiments, the        presence of 2′/3′-O-acetyl-ADP-ribose indicates the presence of        Sir2 activity in the composition. These methods can be used with        any acetylated peptide substrates, including radiolabeled        substrates, previously described.

Additional embodiments of the invention are directed to methods ofdeacetylating an acetylated peptide. The methods comprise combining thepeptide with a Sir2 enzyme. In novel aspects of these embodiments, theacetylated peptide is not a histone. As with previously describedmethods, any Sir2 enzyme that produces 2′/3′-O-acetyl-ADP-ribose isuseful for these methods; the methods would also be expected to beuseful for the deacetylation of any acetylated peptide that consists ofat least two amino acids, wherein at least one of the amino acidscomprises a lysine residue that is acetylated at the ε-amino moiety.

The invention is also directed to methods of inhibiting thedeacetylation of an acetylated peptide. The methods comprise mixing theacetylated peptide with a Sir2 inhibitor determined by the methodspreviously described. These methods are useful for inhibitingdeacetylation of any acetylated peptide in vitro or in vivo. Forexample, the acetylated peptide can be in a living cell, e.g., amammalian cell, which can optionally be a cell in a living mammal, suchas a human. A useful embodiment is inhibiting deacetylation of p53 in amammal at risk for, or suffering from, cancer induced by lack of p53-DNA binding.

As is well known, products of enzyme reactions are generally inhibitorsof the same enzyme. Such is also known to be true with Sir2, wherenicotinamide inhibits the enzyme⁴⁶. We have also shown that ADPR is apotent inhibitor of AfSir2 (see Example 1). Thus, it would also beexpected that 2′/3′- O-acetyl-ADP-ribose would inhibit Sir2.Accordingly, the present invention is directed to methods of inhibitingthe Sir2-directed deacetylation of an acetylated peptide. The methodscomprise mixing the acetylated peptide with 2′/3′-O-acetyl-ADP-ribose orany of the analogs of 2′/3′-O-acetyl-ADP-ribose previously described.Preferred peptides for these methods include histones and p53. Althoughthese methods are useful in vitro, in preferred embodiments theacetylated peptide is in a living cell. The living cell can be aprokaryotic cell or, preferably, a eukaryotic cell. The eukaryotic cellcan be a mammalian cell, optionally in a living mammal, such as a human.Usefully, the mammal is at risk for, or suffering from, cancer inducedby lack of p53 -DNA binding.

Additionally, the invention is directed to prodrugs of2′/3′-O-acetyl-ADP-ribose. The prodrugs comprise2′/3′-O-acetyl-ADP-ribose covalently bonded to a moiety by anesterase-sensitive bond. In these embodiments, the prodrug is capable ofpassing into a cell more easily than 2′/3′-O-acetyl-ADP-ribose. Manymethods for making such prodrugs are known. One example is methods usingsalicylate as a moiety, e.g., using methods described in Khamnei &Torrence⁴⁷.

Preferred embodiments of the invention are described in the followingexamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1 Studies Relating to Products, Mechanisms, and Substrates ofSir2 Methods

Enzyme purifications and assays. Sir2 enzymes were purified as GSTfusions except for Sir2Af1 and Af2 which were purified as nativeenzymes¹⁹. Assays for activity on [³H]-acetylated chicken histones werecarried out as described¹⁹. Peptides were synthesized by the JohnsHopkins Medical Institutions Sequencing and Synthesis Facility andpurified by HPLC on an RP-18 column before use. Peptides JB11 and JB12were assayed for deacetylation as follows. Peptide JB12D (deacetylversion) was made as a standard and comigrated with the JB12 productpeak on HPLC. For peptides JB11 and JB12, 100 μg of peptide wasincubated with 10 μg of GST-Sir2p and 200 mM NAD overnight at 30° C. in50 mM Tris-Cl pH 8.0, 50 mM NaCl. Reaction products were separated onStar chromatography workstation with a C-18 analytical column and a0–100% gradient of acetonitrile. The flow rate was 0.4 ml/min;absorption was monitored at 210 nm. The same reaction conditions wereused for Af2Sir2, TmSir2 and GST-Sir2A except incubation was performedat 55° C. for Af2Sir2 and TmSir2 and at 37° C. for GST-Sir2A. HPLCfractions (500 μl) collected after HPLC separation were analyzed byMALDI-TOF spectrometry on an Applied Biosystems Voyager DE-STRinstrument.

Purification of GST-p300 and reactions with p53. GST-p300 was purifiedfrom plasmid pGEX-2T-p300²⁹, the kind gift of Shelley Berger. Bacterialcells containing expression vector were induced with 300 μM IPTGOD₆₀₀=0.6 and grown at 30° C. for 6 h. Cells were collected and proteinpurified using glutathione-Sepharose4B beads (Amersham-Pharmacia)according to the manufacturer's instructions.

Acetyl-p53 was prepared from yeast strain YPH500 transformed withexpression vector pRB16 containing p53 cDNA and cell extractsprepared^(1,12). Extract containing 4.6 mg of protein was incubated with12 μg of anti-p53 antibody DO-1 conjugated to agarose (Santa CruzBiotechnology) for 1 hour at 4° C. The beads were washed and incubatedwith 5 μg of GST-CBP and 37.5 ml of [³H]-acetyl-CoA (3.7 Ci/mmol, 67 μM,Amersham-Pharmacia) in HAT buffer (50 mM Tris-Cl pH 8.0, 10% glycerol,0.1 mM EDTA, 1 mM DTT, 50 mM NaCl and 4 mM sodium-butyrate) for 1 hourat 30° C. Following acetylation, beads were washed and divided into fourtubes. each containing 10 μl, and incubated for 3 hours with 5 μg ofGST-Sir2A (37° C.), GST-Sir2 (30° C.) and Af2Sir2 (55° C.), 200 μM NAD,50 mM Tris-Cl pH 8.0 and 50 mM NaCl in a final volume of 30 μl. Thecontrol reaction was without enzyme at 37° C. Following deacetylation,beads were washed, and protein released by addition of SDS sample bufferand boiling. The samples were separated on 10% SDS-PAGE. Afterelectrophoresis, the samples were transferred to a PVDF membrane andimmunoblotting was performed using 06-758 antibody (UpstateBiotechnology) or DO-1 according to the manufacturers' instructions.

Methods of Analysis. Reagents and buffers were obtained from commercialventors. HPLC separations of NAD and its products were run in a Waters600 pump/controller, fitted with a Waters 2487 dual wavelength UV/Visdetector or a Waters 996 PDA multi-wavelength detector instrument. HPLCchromatograms were collected with a 260 nm absorbance unless otherwiseindicated. Kinetic studies via HPLC were performed using a programmableWaters 717 autosampler with temperature control. ¹H NMR studies wereperformed on 300 MHz or 600 MHz Bruker spectrometers with computerizedtemperature control. The MS and MS/MS experiments were conducted usingeither a Perseptive Mariner ESI-TOF coupled to a capillary MicroTechC-18 column, or a Finnigan LCQ instrument.

Synthesis of β-1′-AADPR. CD38 was expressed and purified as reported⁴³.100 mg of NAD⁺ was dissolved in 5 mL 4 M NaOAc pH 6.5. To this solutionwas added 50 μg CD38 enzyme dissolved in 50 mM NaOAc pH 5.0. Afterovernight incubation at 37° C. the material was filtered (MilliporeBiomax 10K NMWL centrifugal filter device), injected onto a Waters C-18preparative column of dimensions 19 mm×300 mm, and eluted with 0.5% TFAat a flow-rate of 4 mL/min. A peak eluting before NAD⁺ (40 min versus 50min respectively) was collected and lyophilized. The product (yield—28%)was characterized by MS and NMR and determined to be β-1′-AADPR. Data:¹H NMR D₂O d (8.61, s,1H), (8.38, s, 1H), (6.14, d, 1H ), (5.86 s, 1H),(4.5, m, 1H), (4.35, m, 1H) , (4.18–4.00, m, 6H), (2.02, s, 3H). MS(negative ion) 600 amu. MS/MS (600): 540, 352 and 333 amu.

Purification of 3′ -AADPR and NMR characterization. Sir2Af2 products forNMR analysis were obtained by incubation (40° C. overnight) of 300 μgSir2Af2 enzyme, 20 mg NAD⁺, and 70 mg JB12 peptide in 5 mL 40 mMpotassium phosphate buffer pH 6.25. The reaction was quenched byfiltration through a 10,000 MW cutoff membrane (Millipore Biomax 10KNMWL centrifugal filter device). Fractions of the filtrate (1 mL) wereloaded onto DEAE A-50 columns (1 mL) pre-equilibrated with 25 mM NaOAcpH 6.0. Elution with 2 mL of 25 mM NaOAc pH 6.0, 3 mL 100 mM NaOAc pH6.0 and 2 mL 1M NaOAc pH 6.0 gave fractionation of products. The lastthree 1 mL fractions contained 60%–100% of the acetyl-O-ADP-ribose, asshown by MS and HPLC. Separation of 2′-AADPR and 3′-AADPR wasaccomplished using a semi-preparative C-18 column eluted with 1 mMpotassium phosphate (pH 5.0) at a flow rate of 2 mL/min (5 mL injectionvolume). The largest peak (3′-AADPR) eluted at 14 min and wasroto-evaporated to dryness at 2° C. in less than 30 min under highvacuum. An HPLC chromatogram of this material (50 mM ammonium acetateeluant with a semi-preparative C-18 column of dimensions 7.8 mm×300 mm)indicated it was 95% pure. This material was kept ice-chilled in D₂O and¹H NMRs were taken with probe temperatures varying from 0 to 25° C. 1Dand 2D COESY and NOESY experiments were performed to provide unequivocalassignment of 3′-AADPR, and the time-dependent conversion of thismaterial to the 2′ isomer was observed. 3′-AADPR ¹H NMR, D₂O, δ (8.52,s, 1H), (8.22, s, 1H), (6.09, d, 1H), (5.32, d, 1H beta isomer) (5.17,d, 1H alpha isomer), (5.02, m, 1H), (4.7, m, 1H), (4.45, m, 1H), (4.32,m, 1H), (4.23, m,1H),(4.14, m, 2H), (4.126, m, 1H), (3.95, m, 2H),(2.05, s, 3H). NOEs (2.05, 5.02), COESY cross peaks (5.02, 4.23), (5.32,4.23), (5.17, 4.23). MS (negative ion mode): 600, 540 and 346 amu.

Purification of 2′-AADPR and NMR characterization. The 2′-AADPR isomereluted as the second peak in the HPLC chromatogram described above. HPLC(50 mM ammonium acetate pH 5.0) showed that 2′-AADPR converts readily tothe 3′-AADPR. The 2′-AADPR was characterized by allowing the 3′-AADPR toreach equilibrium with it, at a 47:67 ratio (2′-AADPR:3′-AADPR) asdetermined by integrations of HPLC peaks. The COESY and NOESY crosspeaks and the ¹H NMR data allowed full characterization: 2′-AADPR. ¹HNMR, D₂O, δ (8.52, s, 1H), (8.22, s, 1H), (6.09, d, 1H), (5.39, d, 1Hbeta isomer) (5.20, s, 1H alpha isomer), (4.87, m, 1H), (4.7, m, 1H),(4.45, m, 1H), (4.32, m, 1H), (4.23, m, 1H),(4.14, m, 2H), (4.126, m,1H), (3.95, m, 2H), (2.12, d, 3H). NOEs (2.12, 4.87), COESY cross peaks(4.02, 4.87), (5.39, 4.87), (5.20, 4.87). MS (negative ion mode): 600,540, 346.

Interconversion rates of 2′- and 3′-AADPR. Sample of both 2′- and3′-AADPR were incubated at 15° C. and analyzed by HPLC at 40 minintervals (C-18 column; 50 mM ammonium acetate pH 5.0). The percent ofthe 3′-isomer at a given time was fit to the equation 3′-AADPR (%)=P_(a)exp(−kt)+P_(f) where P_(a)+P_(f)=P₀; where P_(a) is the percent of the3′- isomer above equilibrium at time=0, t is time and k is the firstorder rate constant for the conversion. P_(f) is the percent atequilibrium and P₀ is the initial percent. HPLC and NMR analysisestablished the 2′- and 3′-isomers concentration as a function of time.NMR was in 10% d₄-methanol/90% D₂O at 0° C. or in 100% D₂O at 20° C.

Low temperature studies of Sir2Af2 to identify order of productproduction. Sir2Af2 catalyzed deacetylation reactions were performed at8° C. to determine the order of product formation. A 200 μL solutioncontaining 150 μM NAD⁺, 300 μM peptide JB12, in 40 mM potassiumphosphate pH 6.2 were cooled to 8° C. and then 1 μL of 9 μg/μL ofSir2Af2 was added to initiate the reaction. Reactions were assayed byHPLC using 0.5% TFA as an eluant. Peals at 17 and 20 min were used toquantitate 3′- and 2′-AADPR at 30 minute intervals.

Low temperature ¹H NMR analysis of Sir2p reaction products. A solutionof 1.7 mg Sir2Af2 enzyme in 95% D₂O, 50 mM potassium phosphate pH 6.3was concentrated to 550 μL using centrifugation filtration. This samplewas mixed with 2 mg JB12 peptide and cooled to 5° C. in a 600 MHz NMRspectrometer. Spectra were obtained and 280 μL of ice-chilled 22 mM NAD⁺solution in 50 mM potassium phosphate pH 6.3 in 99% D₂O, was added toinitiate the reaction. ¹H NMR 1D spectra were obtained at intervals over18 hr at 50° C.

Elimination of β-1′-AADPR as a Sir2p product. Authentic β-1′-AADPR (100μM, in 40 mM phosphate buffer at pH 6.25 was incubated for one hr at 55°C., and analyzed by HPLC for production of 2′- or 3′-AADPR isomers. Theresulting chromatograms showed no change in the β-1′-AADPR and noproduction of 2′- or 3′-AADPR as compared to controls.

H₂ ¹⁸O incorporation into 2′-and 3′-AADPR. Reactions containing 2 μgSir2Af2 in 200 μL H₂ ¹⁸O (94.5%, containing 40 mM potassium phosphate pH7.0), and 100 μg JB12 peptide and 300 mM NAD⁺ were incubated at 55° C.and assayed by injection onto a C-18 capillary column in LCMS negativeion mode, with 0.5% formic acid as eluant. Peptide, NAD⁺, ADPR, andAADPR were well resolved. MS of ¹⁸O incubations versus ¹⁶O controlsindicated a 2 amu mass increase (602 versus 600) for AADPR, due to asingle ¹⁸O incorporation. MS/MS of both ¹⁶O and ¹⁸O samples wereidentical with a major fragment observed at m/z=540 due to loss ofHOOCCH₃, and establishing that ¹⁸O is incorporated into the acetategroup. Purification of 2′- and 3′-AADPR from ¹⁸O reactions establishedthat both incorporate a single ¹⁸O.

Acid-catalyzed ¹⁸O exchange reactions. Sir2 reactions containing eitherH₂ ¹⁶O or H₂ ¹⁸O (as above) were quenched with 2 volumes of H₂ ¹⁶O or H₂¹⁸O in 10% d₄-acetic acid. Analysis by LCMS (as above) and MS/MS(molecular mass and fragmentations) gave: (¹⁶O, Sir2Af2 reaction, ¹⁸O,H⁺) M⁻ =602, fragmentation=542; (¹⁸O, Sir2p reaction ¹⁸O, H⁺) M⁻604,fragmentations=542, 540; (¹⁸O, Sir2p reaction, ¹⁶O, H⁺) M⁻=602,fragmentation=540; (¹⁶O, Sir2p reaction, ¹⁶O, H⁺) M⁻=600,fragmentation=540. An ADPR sample was incubated in the quench medium asa control with results as follows: (¹⁶O, H⁺) M⁻=558 ; (¹⁶O, pH 7.5)M⁻=558; (¹⁸O, H⁺) M⁻=560; (¹⁸O, pH 7.5) M⁻=558. Similar results wereobtained with yeast Sir2p.

Ara-F-NAD⁺ as an NAD⁺ analogue. Ara-F NAD⁺ is an inhibitor ofADP-ribosyl-transferase and was used to evaluate Sir2Af2. Ara-F-NAD⁺ wassynthesized as previously reported⁴⁴. Deacetylation of peptide JB12 (300μM) by Sir2Af2 (10 μg) in the presence of ara-F-NAD⁺ (50 μM) was testedin 200 μL of 40 mM potassium phosphate pH 6.25. No change of ara-F-NAD⁺was detected by C-18 reverse phase HPLC. Incubation of ara-F-NAD⁺ with 5mM [Carbonyl-¹⁴C]-nicotinamide (55 mCi/mmol; American radiolabeledchemicals) in the presence of 300 μM peptide and 10 μSir2Af2 (20 μL)followed by HPLC analysis indicated no radioactivity incorporated intoara-F-NAD⁺. A control run with JB12-[Carbonyl-¹⁴C]-peptide and NAD⁺established [Carbonyl-¹⁴C]-nicotinamide exchange into NAD⁺, confirmingthe Sir2 base-exchange reaction. A second control used theADP-ribosyl-transferase CD38 as the exchange enzyme, showing full baseexchange between [¹⁴C]nicotinamide and ara-F-NAD⁺ . Inhibition ofSir2Af2 by ara-F-NAD⁺ was evaluated by incubation of Sir2Af2 enzyme (7μM) and ara-F-NAD⁺ (20 μM) in the presence of 300 μM JB12 peptide inpotassium phosphate pH 6.25 buffer. A reaction mixture lacking ara-FNAD⁺ was the control. A positive control used the same reactioncomponents but 500 nM CD38 in the presence of 5 μM ara-F-NAD⁺. Reactionswere initiated after 6 h by adding 150 μM NAD⁺. Reactions were evaluatedfor catalytic turnover using HPLC. Catalytic turnover of Sir2Af2 was notinhibited by ara-F-NAD⁺ under these reaction conditions.

Results

Acetyl p53 and fragments are substrates of Sir2 deacetylases. Histonesare proposed to be a major substrate for Sir2 protein in yeast and otherorganisms. However, many proteins are acetylated at the ε-amino groupsof lysines²⁴. A well-characterized example is p53, acetylated on lysines373 and 382. Synthetic 18-mer peptides, corresponding to p53 fragmentsacetylated at those positions, were incubated with Sir2 enzymes. Allenzymes tested, except Sir2Af1, and including Sir2Af2, human Sir2A andyeast Sir2, deacetylated the peptides acetylated on residue 373 (JB11)and 382 (JB12) (FIG. 1A; Table 1). Further experiments indicated thatJB12 was a >4-fold better substrate for Sir2Af2 than JB11 (FIGS. 1B,1F). As with deacetylation of histones and histone-derived peptides, p53peptide deacetylation was absolutely dependent on NAD⁺. Deacetylationwas directly confirmed by separation of substrate and product by HPLCover C-18 columns (FIGS. 1B, E–G) and MS determination of masses ofsubstrate and product peaks (FIGS. 1C, D, F inset).

Sir2Af2 and other Sir2-like enzymes were also tested for activity onfull-length native acetylated p53. Native acetylated p53 was obtained byacetylating immunoprecipitated p53 in vitro with p300. Theimmunoprecipitation used monoclonal antibody DO-1, raised against theN-terminus of p53. It precipitates both acetylated and nonacetylatedforms of p53. Acetylated p53 was incubated in the presence and absenceof Sir2 enzymes (FIG. 1H). The proteins were separated on SDS-PAGE gelsand immunoblotted with an antibody specific for p53 acetylated atpositions 373 and 382 as well as with FL-393, an antibody that binds toall forms of p53. All three Sir2p enzymess tested deacetylated p53 (FIG.1H).

TABLE 1 Activity of various Sir2 enzymes on histone and p53 substrates.Substrate and Products NAD⁺ [³H]- p53 products Enzyme Organism histones(native) JB11 JB12 analyzed^(a) Sir2p Saccharomyes + + + + + cerevisiaeSir2Af1 Archaeoglobus − −  NT^(b) − NT fulgidus Sir2Af2Archaeoglobus + + + + + fulgidus Sir2Tm Thermotoga + NT + + + maritimacobB Salmonella + NT + NT + typhimurium Sir2A Homo sapiens + + + NT +^(a)AADPR and nicotinamide products were observed by HPLC/MS ^(b)NT, nottested

Activities and reaction products are conserved with several Sir-2-likedeacetylases. The p53 deacetylase activity of the Sir2 enzymes tested(yeast Sir2p, Sir2Af1 and 2, Sir2Tm, cobB and human Sir2A) isphylogenetically conserved. The enzymes fall into two classes: (1) thepredominant class of proteins active on all physiologic substratestested (intact histones, native p53, peptides JB11 and JB12), and (2)the exception, Sir2Af1, reportedly active on chemically acetylatedp53¹⁸, but inactive on all substrates tested here.

The products of the reactions observed by HPLC include nicotinamide,ADP-ribose and AADPR in reactions catalyzed by purified enzymes fromarchaeal Sir2Af, human Sir2A, yeast Sir2p, and eubacterial cobB andSir2Tm proteins. AADPR was formed in all cases, as well as varyingamounts of ADP-ribose.

Chemical synthesis of β-1′-AADPR. The AADPR product of the Sir2 reactionhas the mass expected for β-1′-AADPR, but the same mass is expected for2′-AADPR and 3′-AADPR^(15,16). We synthesized authentic β-1′-AADPR by avariation of a previously reported method²⁵. The retainingglycohydrolase CD38 formed milligram quantities of the β-1′-AADPR isomerwhen incubated with millimolar concentrations of NAD⁺ in saturated (4 M)sodium acetate at pH 6.5 (see Scheme 1A of FIG. 8 for synthesis andstructure). HPLC comparison of standard β-1′-AADPR (See NMR in FIG. 2)and AADPR generated by Sir2p showed that the two compounds were differed(retention times 28 and 32 min, respectively). The difference inchemical identity between Sir2p-derived AADPR and β-1′-AADPR wasconfirmed by MS/MS studies, showing that fragmentation patterns of Sir2pproduct and β-1′-AADPR differed although their masses were identical(FIG. 3).

Characterization of 2′- and 3′-AADPR from Sir2p reactions. Products ofthe Sir2p reaction were produced in reactions containing 100 mg JB12peptide, 30 mg NAD⁺ and 300 μg enzyme and purified by anion exchange andHPLC chromatography. Samples dissolved in 750 μL volumes of ice-chilledD₂O were determined to be approximately 95% pure prior to ¹H NMRanalysis.

The ¹H NMR of the Sir2p product (FIG. 2, middle panel) is different fromthat of β-1′-AADPR, although the acetate moiety is present, based uponthe methyl resonance at 2.1 ppm (FIG. 2, top and bottom panels). Whenthis sample was reanalyzed after refrigeration overnight the ¹H NMRspectrum changed. The minor peaks (FIG. 2, middle panel) became morefully developed (FIG. 2, bottom panel); subsequent spectra indicatedthat a stable ratio of peaks had been established. HPLC analysis yieldedtwo peaks absorbing at 260 nm. Analysis by MS and MS/MS confirmed thatboth molecular ions had the predicted AADPR mass of 600 (m/z, negativemode) and both had identical fragmentation patterns (MS/MS), stronglysupporting the products as 2′- and 3′-regioisomers of AADPR (seeMethods).

Identification of 2′- and 3′-AADPR isomers. We identified the twospecies as 2′- and 3′-AADPR isomers on the basis of 2D NMR experiments(COESY and NOESY) on freshly purified AADPR performed at 0° C. in 10%d₄-methanol, 90% D₂O. These spectra demonstrated clearly that the isomerfirst isolated by HPLC and characterized was the 3′-isomer on the basisof a set of complete COESY cross-peaks and a NOE between the acetylmethyl group and the peak assigned to H3′. Conversion of 3′- to2′-isomers reached a 67:47 equilibrium and the 2′-isomer wascharacterized in a sample containing both 2′- and 3′-isomers. The2′-assignment was made with a complete set of COESY cross peaks and aNOE between the acetyl methyl groups and the peak assigned to H2′ (FIG.9).

NMR and HPLC methods demonstrated interconversion of 3′- to 2′-isomersto provide an equilibrium mixture at 15° C. and 20° C., respectively.Stack plots for both methods (FIG. 4) were fit to an exponential decaycurve and allowed determination of the first-order rate constant foracetyl migration k_(3′ to 2′) of 0.32 h⁻¹ (FIG. 4, upper panel). Theequilibrium constant K was found to be 1.4 at 15° C., based upon HPLCpeak integrations of the 2′- and 3′-isomers. The calculated value fork_(3′ to 2′) was thus 0.45 h⁻¹. A full equilibrium and kineticdescription of acetyl migration including the equilibria for theanomeric forms is shown in Scheme 1B of FIG. 8.

Order of production of the 2′- and 3′-isomers. The order of AADPRregioisomer generation by Sir2p was established in an ¹H NMR experimentat 5° C. (see methods). The stacked spectra (FIG. 5) reveal that the 2′isomer builds up early in the reaction followed by production of the 3′isomers. Each peak from the C1′-H α and β forms of the respectiveregioisomers was well resolved. This result establishes that the kineticsequence for AADPR production is 2′ before 3′ (Scheme 1C of FIG. 8). Therate of Sir2p catalysis exceeds the rate constant for interconversionwith a ratio k_(cat)/k_(2′→3′) of 20 at 5° C.

Incorporation of H₂ ¹⁸O into product. Reaction stoichiometry for Sir2p(Scheme 1C of FIG. 8) indicates that two new oxygen atoms areincorporated into AADPR. It has been assumed that one or more of theseoxygen atoms comes from water^(14,16,18). The Sir2p deacetylationreaction can be viewed as a hydrolysis reaction yielding amino lysineand acetate coupled to an ADP-ribosyl transfer reaction where ADPR istransferred to acetate.

Reactions in ¹⁸O water followed by MS analysis of the AADPR productindicated that product mass is increased by 2 amu consistent withincorporation of a single ¹⁸O from water into AADPR. The ¹⁸O enrichment(65%) was the same as the initial ¹⁸O content in the water assay (FIG.5, inset). Dilution of the samples in 2 vol of ¹⁶O water at pH 7.5 or in10% d₄-acetic acid followed by MS analysis (time range 30 min-24 h)showed that ¹⁸O content did not change (Table 2). The site of ¹⁸Oincorporation is therefore a non-exchangeable oxygen atom in the2′-AADPR and 3′-AADPR molecules.

The ¹⁸O atom could reside in either the acetate moiety of 2′- and3′-AADPR molecules or in the 1′-OH of the product. Incubation ofunlabeled ADPR and 2′- and 3-AADPR in ¹⁸O in 10% d₄-acetic acid forthree h at room temperature increased mass by 2 amu consistent withincorporation of a single ¹⁸O into the molecules, but in neutralconditions no such change in mass was observed. Only the 1′-OH isconsistent with this exchange. 2′- and 3′-AADPR derived from¹⁸O-containing Sir2p reactions when incubated with 10% d₄-acetic acidand ¹⁸O water incorporated a second ¹⁸O into the AADPR molecule(mass=604) consistent with isotope exchange of the 1′-OH anddemonstrating the presence of a free hydroxyl (FIG. 5, inset).

Exchange experiments were combined with MS/MS studies and reinforcedthis chemical interpretation (Table 2). Acid catalyzed exchangereactions of 2′- and 3′-AADPR in ¹⁸O water gave a 542 amu fragment,consistent with incorporation of a single ¹⁸O into the ribose moiety.Reactions of Sir2p run in either ¹⁶O or ¹⁸O water led to 540 amufragments that originate from loss of acetate. Thus, thenon-exchangeable ¹⁸O atom obtained from solvent is incorporated into thecarbonyl oxygen of 2′- and 3′-acetates.

TABLE 2 Mass Spectrometry: observed species in the presence of ¹⁸O and¹⁶O water Reaction Molecular Species Fragmentations conditions Quenchconditions (m/z) (major species) ¹⁶O NQ 600 540, 346 ¹⁸O NQ 602 540, 346¹⁶O ¹⁸O, H⁺ 602 542, 346 ¹⁸O ¹⁸O, H⁺ 604 542, 346 ¹⁸O ¹⁶O, H⁺ 602 540,346 ADPR ¹⁸O, pH 7.5 558 346 ADPR ¹⁸O, H⁺ 560 346 The reactionconditions for the first five entries reflect Sir2Af2 mediateddeacetylation at pH 7.8, 50 mM potassium phosphate at 55° C. for 30 minwith enrichment of the reaction water content (minimal 65%) with theisotope shown. The reaction was subsequently analyzed by LCMS in MS andMS/MS modes. The molecular species reflects the observed major ionidentified at 7 min, the retention time of AADPR in LC. Fragmentationsare based upon MS/MS spectra derived from selection of the majormolecular ion. Not quenched (NQ) reflects injection of otherwiseuntreated reaction mixtures. In cases where a quench was used, 2 volumesof either 95% H₂ ¹⁸O or unlabeled water containing 10% d₄-acetic acidwas added to reaction mixtures after initial 55° C. incubation andreacted for 3 h at room temperature. Samples were subsequently analyzedby LCMS and MS/MS. The bottom two cases reflect behavior of ADPR ineither 50 mM potassium phosphate pH 7.5 or 95% H₂ ¹⁸O containing 10%d₄-acetic acid after incubation for a three hour period at roomtemperature.

Inhibitor ara-F-NAD⁺ substrate reaction, exchange and inhibition.Ara-F-NAD⁺ is a powerful inhibitor for many ADP-ribosyltransferenzymes²⁶, since the substitution of fluorine for OH at the 2′ positionmakes ara-F-NAD⁺ roughly 50 times more stable than NAD⁺²⁶ and thuspermits the chemical trapping of enzyme-ADP-ribose covalentintermediates²⁷. Ara-F NAD⁺ did not serve as a substrate indeacetylation or exchange reactions. Controls readily exchangedradiolabeled nicotinamide (5 mM) into ara-F-NAD⁺ and Sir2p exchangednicotinamide into 50 μM NAD⁺ under these conditions. Ara-F-NAD⁺ did notinactivate Sir2p in the presence or absence of peptide dispite itsactivity on the control using CD38. Thus, ara-F-NAD⁺ is inert in theSir2p reaction.

Discussion

We show here that acetylated human p53 tumor suppressor and peptidesderived from it are excellent in vitro substrates for a wide variety ofSir2ps. Because acetylation is required for activation of efficientsequence-specific DHA binding, deacetylase action is predicted todecrease p53 activity in cells. Previous studies have shown a complexrelationship between p53 and the Rpd3 family of deacetylases. p53 actsas a repressor at certain loci, and the Rpd3 histonedeacetylase-associated factor, Sin3p, mediates such repression37.Paradoxically, one study suggests that p53 itself can be a a substratefor Rpd3 family deacetylases, at least when overexpressed³⁸.Deacetylation of p53 by Sir2ps would allow its activity to be modulatedindirectly via cellular metabolic or redox states. This would makesense, particularly following oxidative stresses, which are associatedwith DNA damage. However, our studies do not indicate any specificityamong Sir2 proteins for p53. Those enzymes that efficiently deacetylatehistones work well on p53 as well—even if they are derived from Archaea.Recent work from other laboratories also shows that acetylated BSA, anon physiologic substrate, is a substrate for Sir2afl¹⁸. Thus, if Sir2psplay regulatory roles in modulating p53 activities, there must beunidentified factors or conditions to make such NAD⁺-regulateddeacetylation reactions specific.

Product of Sir2p. The identification of 2′-AADPR as the product of Sir2pdeacetylation has reinforced the view that Sir2ps represents a uniquetype of NAD⁺-dependent enzyme. The product of Sir2p has been proposed tobe β-1′-AADPR¹⁵⁻¹⁷ and the X-ray crystal structure of Sir2p from Min etal.¹⁸ was interpreted in terms of formation of this species. Thisproduct was suggested from the precedent of other ADP-ribosyltransferases that also catalyze nicotinamide exchange reactions similarto the Sir2p reaction. NAD⁺ glycohydrolases and CD38 both form productswith β-configuration and both catalyze base-exchange reactions. Thus theproposal for the Sir2p product was a logical extension of knownenzymology¹⁵⁻¹⁷. Base exchange requires enzyme stabilization of an ADPRelectrophile, or formation of a covalent enzyme intermediate²⁷, normallyresulting in ADPR transfer to nucleophiles with 1-β-stereochemistry. Wesynthesized authentic β-1′-AADPR and established that none of theproduct formed by Sir2p corresponds to this compound. The stabilityobserved for β-1′-AADPR also establishes that it is not an intermediatein the Sir2p reactions. The product released from the catalytic site ofSir2p is 2′-AADPR which forms a Sir2p-independent mixture of 2′- and3′-acetyl isomers. The chemical exchange of the acetyl group results ina nearly 2′:3′ equilibrium ratio of 67:47 at 15° C. The exchange occursat rates of 0.45 hr⁻¹ and 0.32 hr⁻¹ at this temperature. Atphysiological temperatures of 37° C., the equilibrium will beestablished within 60 min. Thus, it is conceivable that a cascadedownstream of Sir2p action could be timed by the spontaneous conversionof 2′- to 3′-AADPR. Each regioisomer exist in 1′ α and β anomeric forms,generating four distinct species by NMR (Scheme 1B of FIG. 8). The α/βanomeric equilibrium is fast (>50 s⁻¹).

Sir2p catalyzed steps that generate 2′-AADPR. The formation of 2′-AADPRrequires that the 2′-hydroxyl act as a nucleophile at some stage of thechemical mechanism. Participation of the 2′-hydroxyl has been suggested.The 2′-hydroxyl was proposed to act directly as the nucleophile todeacetylate the amide moiety¹⁶. The direct 2′-nucleophile mechanismrequires incorporation of ¹⁸O from solvent water at the 1′-position asthe sole ¹⁸O label in the 2′-AADPR released from Sir2p. However, MSanalysis established that ¹⁸O appears in the acetyl group, thereforeaction of the 2′-hydroxyl group as the primary nucleophile can beeliminated.

To reconcile the coupling of ADPR transfer with deacetylation, thereaction can be compared to the ADP-ribosyltransfer reactions catalyzedby the ADP-ribosylating toxins in which the enzymes generate a highlyreactive ADP-ribooxocarbenium ion that is attacked by a weaknucleophile. For example, in cholera toxin, ADPR is transferred fromNAD⁺ to an Arg with stereochemical inversion of the ADP-ribose togenerate a α-1′-substituted ADPR-amidate³⁹. This same strategy in theSir2p reaction generates the ADP-ribo-oxocarbenium ion that captures theacyl oxygen of the N-acetyl-lysine to generate an O-alkyl-amidate(Scheme ID of FIG. 8). O-Alkyl-amidates are chemically activated andspontaneously hydrolyze in water to form free amines and esters^(40,41).For Sir2p these correspond to the deacetylated lysine and theα-1′-AADPR. Transition state studies of all ADP-ribose transferreactions thus far characterized show that a powerful ribo-oxocarbeniumion electrophile is generated to facilitate ADPR transfer chemistry⁴².

This mechanism explains the requirement of the peptide substrate fornicotinamide exchange; even in mutagenized enzymes that lack deacetylaseactivity¹⁸. There are two possible reaction pathways that can form2′-AADPR. In the first, the intermediate O-alkyl-amidate hydrolyzes to a1′-substituted α-AADPR that isomerizes to 2′- regiochemistry to give theobserved product (Mechanism A of FIG. 7; Scheme 1D of FIG. 8).Alternatively, the 2′-hydroxyl could attack the O-alkyl-amidateintermediate to generate a cyclic acyloxonium, followed by hydrolysis toform the observed 2′-AADPR product (Mechanism B of FIG. 7; Scheme 1E ofFIG. 8).

Nature of the α-1′-AADPR intermediate. Low temperature NMR studiesperformed at 5° C. using Sir2Af2 furnished no evidence for accumulationof α-1′-AADPR. Therefore, conversion of α-1′-AADPR to 2′-AADPR occurs≧the catalytic rate of the enzyme, since the detection level for theα-1′-AADPR was near the enzyme concentration. The 2′- to 3′-AADPRinterconversion is slower than k_(cat) by a factor of 20 andenzyme-dependent production of the 2′-isomer was observed by NMR (FIG.4). The mechanism most consistent with the results is the 2′-hydroxylattack on the 1′-O-alkylamidate to form a 1′-2′ substituted acyl-oxoniumstructure as the precursor to 2′-AADPR formation on the enzyme(Mechanism B of FIG. 7; Scheme 1E of FIG. 8).

Label pattern from H₂ ¹⁸O incorporation. Water was confirmed as aparticipant in the catalytic mechanism by incorporation of a single ¹⁸Oatom from ¹⁸O water in the AADPR product. This oxygen atom is anonexchangeable oxygen of the acetyl group. Confirmation of the singlewater molecule mechanism and of an acid-exchangeable oxygen at theC1′-hydroxyl confirm an acyl-oxygen-C1′ bond formation and the attack ofwater at the carbon of the amide carbonyl.

X-ray structure is consistent with formation of observed products.Initial inspection of the Sir2-Af1 X-ray structure¹⁸ shows that thereare no putative nucleophilic residues within bonding distance of NAD⁺ tosupport a covalent ADPR transfer and a β-stereochemical outcome. A freshview of the structure in light of our experimental results isintriguing, as the a face of the NAD⁺ is unencumbered from attack by abound acetyl and the 2′-hydroxyl is found unligated to any side chain inthe enzyme. Indeed, a modeling study allowed facile docking of the p53C-terminal peptide NMR structure into the crystal structure of Sir2Af1(FIG. 6). In this model, the acetyl oxygen is indeed perfectlypositioned to approach the a face of the nicotinamide bound to Sir2Af1.Furthermore, the histidine located near the 3′ hydroxyl has been shownby mutagenesis to be essential for deacetylase function, suggesting thatthe bottom face of the NAD⁺ molecule is subject to acid-base transferpathways to fulfill catalytic function.

Summary of the Sir2p mechanism. The sum of these results provides strongsupport for the reaction steps of Scheme 1E of FIG. 8. Most histonedeacetylases are simple hydrolases—hydrolysis of N-acetyl groups is achemically simple and energetically favorable reaction. Biologicalrationales for consumption of metabolically valuable NAD⁺ as acosubstrate may include substrate signaling; NAD⁺ levels may signalcellular energetic and redox states to control Sir2p-based generegulation. A second possibility is that the reaction initiates a Sir2psignal transduction pathway by generation of the novel compounds 2′- and3′-AADPR. These molecules have not been previously recognized inmetabolism, and carry the features of chemical instability common toother initiators of signaling pathways. Recent evidence that some Sir2phomologues are cytosoliC^(35,36) and can accept acetyl groups from othernon-histone proteins such as chemically acetylated BSA¹⁸ or p53, suggesta much broader role in cell development and transcriptional regulationby acetyl-transferase reactions. Identification of the products of Sir2pprovides the tools for further investigations of these pathways andprovides information for inhibitors designed sepecifically for thisunusual ADP-ribosyl-transferase.

Example 2 Improved Assays for Sir2 and Inhibitors of SIR2

As discussed above, the reaction of Sir2 with NAD⁺ and an appropriateprotein or peptide acetyl donor produces 2′ and 3′-O-acetyl-ADPR (AADPR)and deacetylated protein or peptide. The detection of Sir2 activity canbe effectively performed by measuring the production of the AADPRproduct. Previous methods for this were based upon the use of HPLC todetect products. By radiochemical labeling of NAD⁺ by known methods theproduction and quantitation of radiolabeled AADPR was achieved by therapid separation of AADPR from unreacted NAD⁺ on Sephadex-DEAE columns.

The method as practiced is as follows: 300 μL of 50 mM potassiumphosphate buffer pH 6.2 (or 7.0 or 7.5) containing 150 μM of the peptideJB12 (p53 based) and 150 mM [5′-¹⁴C]NAD⁺ (specific activity 100000 cpmper sample) were reacted with 3 μg Sir2 enzyme in 5 μL buffer (yeasthomologue Sir2p or Archaeglobus fulgidus AF2Sir2) for a period varyingfrom 30 min to 4 h. Upon completion of reaction time 50 μL aliquots ofreaction mixture were frozen on dry ice. After 4 h all tubes were thawedand loaded onto 1 mL DEAE Sephadex columns pre-equilibrated with 25 mMammonium acetate pH 7.0. 6 mL of 25 mM ammonium acetate was elutedthrough the columns and collected in 1 mL fractions. The column wassubsequently eluted with 6 mL 500 mM ammonium acetate and againcollected in 1 mL fractions. Each column fraction was counted byaddition of 9 mL scintillation fluid and placed into a scintillationcounter. The profiles for the elutions are shown in FIG. 11. Using thismethod, the unreacted NAD⁺ elutes first (fractions 1–6), followed byAADPR (fractions 7–12). The sum of the fractions above 100 cpm weresubtracted with the 0 time counts (FIG. 12). The sensitivity fordetection of AADPR versus NAD is 5 μM in 50 μL, in other words, 250pmoles of AADPR.

This method has numerous applications to the detection of Sir2 activityin biological extracts when combined with controls for the non-Sir2degradation of NAD⁺. We have showed that extracts containing Sir2, whencombined with a suitable acetyl donor substrate, can produce AADPR, andthat activity can be directly measured by formation of AADPR via theabove-described column methodology. Detection of AADPR in bacterialextracts expressing Sir2 activity was performed analogously to thatstated above, except that instead of adding pure enzyme, whole cellhomogenates were added. Controls using uninduced cells, or cells inducedto produce a mutant inactive Sir2 showed no production of AADPR, asdetermined by this assay, and no breakdown of NAD⁺ occurred. Thesensitivity of this assay allows for detections of AADPR that arepresumably near biologically relevant concentrations (5 μM).

The advantages of this method are the speed of the assay, which requiresonly 4–7 hours for up to 20 samples, and its reliance upon directdetection of AADPR, a unique product of the Sir2 reaction. This methodalso permits the facile purification of a pure form of AADPR, as theseparation conceived also functions to separate AADPR from othercomponents in reaction mixtures. Purified AADPR from cellular Sir2action can then be verified in chemical structure by HPLC or MS, or usedin another specific assay.

Further applications of this method include the evaluation of inhibitorsof Sir2 activity. The discovery and quantitative evaluation of theaction of small molecules on Sir2 action has the potential to lead totherapeutic compounds. Utilization of AADPR to detect Sir2 inhibitionrelies upon detecting the sole unique product of Sir2 action in cells.In this method the reaction is performed as described above, with thepotential inhibitor being added in varying concentrations. Reaction ratefor Sir2 is determined in each sample by using the initial linear phaseof reaction progress, which is fastest and is equal to the slope.Inhibitors of the reaction show decreased rates. Inhibition isquantified by the equation1/ν=1/V _(max)(1+[I]/Kii)+K _(m) /V _(max)(1+[I]/Ki)where K_(m) and V_(max) are parameters used to describe the Michaelisconstants of the substrate that is subject to competitive binding withthe inhibitor, [I] is the inhibitor concentration and K_(i) and K_(ii)are the competitive and non-competitive binding constants. This methodwould be expected to discover competitive inhibitors with K_(i) valueswith binding affinities as high as 1 mM, and practically can identifyinhibitors with binding affinities in the range of 100 μM or below. Inone embodiment, the method comprises the addition of 200 μM inhibitor to300 μL reaction mixtures containing 100 μM of NAD⁺ and peptidesubstrate.

Investigations of several extracts, including hamster ovary cells andrat liver has shown that high esterase activity exists that canbreakdown AADPR to ADPR and acetate. The use of AADPR or radiolabeledAADPR is a strategy to characterize and discover the esterases (or otherenzymes) that break down or utilize AADPR in vivo. Specifically, AADPRbreakdown can be measured by HPLC or TLC assay. When radiolabels areused, amounts of AADPR necessary for this purpose can be as small as 250pmoles.

As with the production of AADPR, the use of an AADPR-based method ofdetecting AADPR breakdown furnishes the unique Sir2 reaction product asa means to interrogate the activities that utilize the compound incells. Quantifying breakdown versus time provides a method to discovernot only the AADPR utilizing activities but also compounds that caninhibit this activity. Inhibition of AADPR breakdown in cells by smallmolecule compounds can increase AADPR action in cells, thus extendingSir2 effects, via the increased lifetime of the only unique product ofSir2 action inside of cells.

Esterase activity of DEAE separated rat liver extract as measured bypara-nitrophenol release from para-nitrophenyl-acetate (400 nm) is shownin FIG. 3. This shows the inherent esterase rich background to whichAADPR is subject in liver cells. The use of AADPR as a substrate tointerrogate this broad esterase activity can be used to determine AADPRspecific esterases inside of cells. One strategy is to use the ratio ofAADPR esterase activity/para-nitrophenylacetate esterase activity as ameasure of specificity of the activity for the AADPR product measuredagainst a generic substrate activity. Enzymological theory posits thatenzyme activity is optimized for the substrate for which the enzyme wasdesigned. Thus, activity ratios should be highest for enzymes that aredesigned to recognize AADPR.

Other strategies can also be employed, such as using small moleculeinhibitors that resemble AADPR. As shown in FIG. 13, the esterases aresensitive to mM concentration additions of AADPR analogs.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

1. A purified mixture of 2′/3′-O-acetyl-ADP-ribose in equilibrium.
 2. Ananalog of 2′-O-acetyl-ADP-ribose or 3′-O-acetyl-ADP-ribose exhibitingincreased stability in cells, wherein the 2′-O-acetyl-ADP-ribose and the3′-O-acetyl-ADP-ribose both comprise an acetate group with an esteroxygen, and an oxygen between two phosphorous atoms.
 3. The analog ofclaim 2, wherein a CF₂, a NH, or an S replaces the ester oxygen or theoxygen between two phosphorous atoms.
 4. The analog of claim 3, whereina CF₂ or NH, or a S independently replaces the ester oxygen and theoxygen between two phosphorous atoms.
 5. The analog of claim 3, whereina CF₂, a NH, or an S replaces the ester oxygen.
 6. The analog of claim3, wherein a CF₂ replaces the ester oxygen or the oxygen between twophosphorous atoms, or wherein a CF₂ replaces both the ester oxygen andthe oxygen between two phosphorous atoms.
 7. The analog of claim 3,wherein a CF₂ replaces the ester oxygen.
 8. The mixture of claim 1 in anaqueous composition having a solute, excluding salts, that is at least50% 2′/3′-O-acetyl-ADP-ribose in equilibrium.
 9. The mixture of claim 1in an aqueous composition having a solute, excluding salts, that is atleast 75% 2′/3′-O-acetyl-ADP-ribose in equilibrium.
 10. The mixture ofclaim 1 in an aqueous composition having a solute, excluding salts, thatis at least 90% 2′/3′-O-acetyl-ADP-ribose in equilibrium.